Combining different design strategies for rational affinity maturation of the MICA‐NKG2D interface

We redesigned residues on the surface of MICA, a protein that binds the homodimeric immunoreceptor NKG2D, to increase binding affinity with a series of rational, incremental changes. A fixed‐backbone RosettaDesign protocol scored a set of initial mutations, which we tested by surface plasmon resonan...

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Bibliographic Details
Published in:	Protein science Vol. 21; no. 9; pp. 1396 - 1402
Main Authors:	Henager, Samuel H., Hale, Melissa A., Maurice, Nicholas J., Dunnington, Erin C., Swanson, Carter J., Peterson, Megan J., Ban, Joseph J., Culpepper, David J., Davies, Luke D., Sanders, Lisa K., McFarland, Benjamin J.
Format:	Journal Article
Language:	English
Published:	Hoboken Wiley Subscription Services, Inc., A Wiley Company 01-09-2012 Wiley Subscription Services, Inc
Subjects:	additivity Computational Biology - methods For the Record free energy of binding Histocompatibility Antigens Class I - chemistry Histocompatibility Antigens Class I - genetics Histocompatibility Antigens Class I - metabolism Humans immunoreceptors Models, Molecular Mutation NK Cell Lectin-Like Receptor Subfamily K - metabolism Protein Binding protein design Protein Engineering - methods Protein Interaction Maps protein–protein interaction Recombinant Proteins - chemistry Recombinant Proteins - genetics Recombinant Proteins - metabolism Thermodynamics thermodynamics and kinetics of binding van't Hoff enthalpy
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Summary:	We redesigned residues on the surface of MICA, a protein that binds the homodimeric immunoreceptor NKG2D, to increase binding affinity with a series of rational, incremental changes. A fixed‐backbone RosettaDesign protocol scored a set of initial mutations, which we tested by surface plasmon resonance for thermodynamics and kinetics of NKG2D binding, both singly and in combination. We combined the best four mutations at the surface with three affinity‐enhancing mutations below the binding interface found with a previous design strategy. After curating design scores with three cross‐validated tests, we found a linear relationship between free energy of binding and design score, and to a lesser extent, enthalpy and design score. Multiple mutants bound with substantial subadditivity, but in at least one case full additivity was observed when combining distant mutations. Altogether, combining the best mutations from the two strategies into a septuple mutant enhanced affinity by 50‐fold, to 50 nM, demonstrating a simple, effective protocol for affinity enhancement.
Bibliography:	S.H.H. designed and completed protein biochemistry and computational experiments and edited the manuscript; M.A.H., N.J.M., E.C.D., C.J.S., M.J.P., J.J.B., L.D.D., and L.K.S. designed and completed DNA mutagenesis, protein expression, purification, assessment, and data analysis and edited the manuscript; D.J.C. designed and completed computational experiments and edited the manuscript; and B.J.M.: conceived and designed the experiments, completed computational experiments, and wrote the manuscript. Author contributions ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 23 Grant sponsor: NIH; Grant number: 2 R15 AI058972-02 (to B.J.M.); Grant sponsor: Seattle Pacific University Montana Endowment Grant (to B.J.M.). Megan J. Peterson's current address is the Institute of Molecular Biology, University of Oregon, Eugene, OR 97403. Carter J. Swanson's current address is the Department of Biophysics, University of Michigan, Ann Arbor, MI 48109. Lisa K. Sanders's current address is the School of Medicine, University of Washington, Seattle, WA 98195. Author contributions: S.H.H. designed and completed protein biochemistry and computational experiments and edited the manuscript; M.A.H., N.J.M., E.C.D., C.J.S., M.J.P., J.J.B., L.D.D., and L.K.S. designed and completed DNA mutagenesis, protein expression, purification, assessment, and data analysis and edited the manuscript; D.J.C. designed and completed computational experiments and edited the manuscript; and B.J.M.: conceived and designed the experiments, completed computational experiments, and wrote the manuscript.
ISSN:	0961-8368 1469-896X
DOI:	10.1002/pro.2115